Bisaryl hydrazones as exchangeable biocompatible linkers

Article abstract of DOI:10.1002/anie.200906756

(Figure Presented) Catch and release: Stable bisaryl hydrazones undergo rapid exchange under mild conditions in the presence of a catalyst (see schematic illustration; b=biotin). This highly efficient hydrazone exchange is useful for the affinity purifica

Full text of DOI:10.1002/anie.200906756

Angewandte
Chemie
DOI: 10.1002/anie.200906756
Cleavable Linkers
Bisaryl Hydrazones as Exchangeable Biocompatible Linkers**
Anouk Dirksen, Subramanian Yegneswaran, and Philip E. Dawson*
The selective enrichment of tagged molecules from complex
biological mixtures is of primary importance in chemical
biology and proteomics. One of the most widely applied
enrichment methods is affinity purification on (strept)avidin
beads with biotin as an affinity tag. This strategy takes full
advantage of the high affinity of biotin for (strept)avidin (Ka
ꢀ 1.7 ꢀ 1015 mÀ1).[1] However, the method is limited by the
harsh, denaturing conditions required for elution, such as
boiling in a buffer containing sodium dodecyl sulfate (SDS) or
treatment with an 8m solution of guanidine (pH 1.5). Under
these conditions, protein structure and function are lost, and
target proteins may be contaminated with proteins bound
nonspecifically to the beads. Two strategies have been
explored for elution under mild conditions: 1) weakening of
the biotin–(strept)avidin interaction by modulation of the
Ka value[2] and 2) introduction of a proteolytically[3] or chemi-
cally[4] cleavable linker. Although the first strategy does
improve the release of biotinylated proteins from (strept)a-
vidin beads, it adversely affects the stringency of the
immobilization. The second strategy enables site-specific
cleavage; however, premature cleavage has been reported,
and cleavage conditions have no demonstrated compatibility
with active biomolecules. Furthermore, the general applic-
ability of the cleavable linkers is often limited by the need for
multistep organic synthesis before implementation.
Herein, we report that stable, yet exchangeable, bisaryl
hydrazone linkers can be cleaved under mild conditions by
catalytic transimination (Figure 1). The mechanism of tran-
Figure 1. Three distinct strategies for the cleavage of a hydrazone
linker: A) capping of the protein, B) label exchange for further analysis
or purification, and C) capping of the beads to leave a synthetic handle
on the protein for further modification.
simination[9] enables the introduction of a new label to trace
the originally biotinylated protein, or a new affinity tag for
further purification (Figure 1B). Alternatively, the biotin
fragment bound to the beads can be capped with a benzalde-
hyde derivative to elute a protein with a synthetic handle that
can be modified at a later time (Figure 1C).
The bisaryl hydrazone formed between a benzaldehyde
and a hydrazinopyridine group has been successfully applied
as a stable linker in bioconjugation and biomolecular label-
ing.[3a,10,11d] Notably, it has been included as a stable compo-
nent of biotinylated protease-cleavable linker[3a] and as a
stable chromogenic biotinylation agent.[10] The nucleophilic
catalyst aniline accelerates effectively hydrazone formation
and hydrolysis,[11] and an enhancement of the rate constants
by two orders of magnitude is possible without a change in the
pH value.[11d] As a result, the stable bisaryl hydrazone will
become dynamic upon addition of the catalyst, which will
make the bond prone to exchange and cleavage.
Indeed, a mixture of two bisaryl hydrazones (100 mm each)
was stable at pH 7.0 (> 22 h), but rapidly reequilibrated in the
presence of 100 mm aniline at pH 7.0 (< 8 h) to give four
hydrazones (50 mm each; see Figure 1 in the Supporting
Information). Reequilibration proceeded with the rate con-
stant of catalyzed hydrolysis, which suggests that scrambling
occurs through a direct reaction of trace free hydrazine and
free aldehyde present in solution at equilibrium. Surprisingly,
despite considerable analysis of the dynamic covalent
chemistry of hydrazones,[12] hydrazone reequilibration under
physiological conditions in the absence of excess monomers
has not been reported previously.
Hydrazones have been explored for the reversible con-
jugation[5] and labeling[6–8] of biomolecules. However, their
application has been limited, as most hydrazones are prone to
hydrolysis and premature cleavage, whereas hydrazones (and
oximes) that are fully stable under biological and mildly acidic
conditions undergo slower hydrolysis and are difficult to
exchange or cleave. In principle, a molecular catalyst could be
used to accelerate hydrolysis and enable an effective tran-
sition between a stable state for workup and a dynamic state
for cleavage and exchange.
[*] Dr. A. Dirksen, Dr. P. E. Dawson
Departments of Cell Biology and Chemistry
The Scripps Research Institute
10550 North Torrey Pines Rd, La Jolla, CA 92037 (USA)
Fax: (+1)858-784-7319
E-mail: dawson@scripps.edu
Dr. S. Yegneswaran
Department of Molecular and Experimental Medicine
The Scripps Research Institute
10550 North Torrey Pines Rd, La Jolla, CA 92037 (USA)
[**] We thank Prof. John H. Griffin for providing resources, and
Phuong M. Nguyen (TSRI) for technical support. This research was
supported by NRSA (HL 07695-15 (S.Y.)) and NIH (GM059380
(P.E.D.)). P.E.D. is a consultant for SoluLink Biosciences Inc. USA.
The ability to convert the hydrazone bond effectively
between stable and dynamic states is key for its use as an
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200906756.
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proteins. To gain insight into the
cleavage efficiency on beads, we per-
formed a model protein study in
which biotinylated human serum
albumin, HSA-CLB354x (8 mm; x is
the average number of CLB354 mol-
ecules per HSA molecule), was
retrieved from a well-defined protein
mixture consisting of aprotinin, cyto-
chrome c, myoglobin, and aldolase
(10 mm each) in a solution at pH 7.4
containing
sodium
phosphate
(50 mm) and NaCl (150 mm). HSA-
CLB354x (x = 1.3) was pulled down
from the protein mixture with avidin
beads. The beads were washed and
incubated for 2 h at pH 6.0 with
varying amounts of NH2OH and ani-
line (Figure 3) to elute HSA by cap-
ping the protein (Figure 1A). As
reflected by SDS-PAGE analysis, the
cleavage efficiency was improved, in
accordance with the solution study, by
increasing the concentration of
NH2OH as well as by the addition of
aniline.
Figure 2. a) Transimination of biotinylation agent CLB354 with aniline in the presence of
NH2OH.[10] b) Graph showing the concentration of CLB354 (initial concentration: 5 mm) in sodium
phosphate buffer (0.1m, pH 6.0) with respect to time in the absence of amines (stable), in the
presence of aniline (100 mm, reequilibration), and in the presence of NH2OH (100 mm) and
aniline (100 mm, capture).
A hydrazone can be cleaved by
transimination with any aminooxy
exchangeable linker in pull-down assays for the enrichment
and purification of proteins. The biotinylation agent CLB354
(Figure 2a) is commercially available, and its hydrazone
linker has a distinct absorption in the visible region.[10] The
reversibility and stability of CLB354 were first validated in
solution at pH 6.0 to facilitate both reequilibration kinetics
and protein stability. The CLB354 hydrazone (5 mm) was
found to be fully stable overnight; however, upon treatment
with 100 mm aniline, approximately 25% of the hydrazone
was hydrolyzed rapidly, and a new equilibrium was attained
(Figure 2b). As the hydrazone has a Keq value of approx-
imately 106 mÀ1 in aqueous buffer,[11d] it would need to be
diluted significantly to shift its equilibrium toward the starting
materials and disrupt the hydrazone bond. However, the
equilibrium can be pushed toward cleavage by trapping the
free aldehyde with an aminooxy compound, such as hydroxyl-
compound. This approach offers the possibility to refunction-
alize HSA by exchanging the biotin tag with a new label or
affinity tag (Figure 1B). To demonstrate this principle, we
cleaved the hydrazone of immobilized HSA-CLB354x (x =
1.3) in a second experiment by using a cleavage buffer
containing 10 mm aminooxyacetyl-Alexa Fluor 488 or amino-
oxyacetyl-modified FLAG peptide and 100 mm aniline
(Figure 3). As reference experiments, the hydrazone was
cleaved with 100 mm NH2OH in the presence and absence of
100 mm aniline (see the Supporting Information), by incuba-
tion with just the cleavage buffer (0.1m sodium phosphate,
pH 6.0), and by boiling in SDS-containing buffer (Figure 3).
Equal amounts of beads were used in each experiment. The
beads were incubated overnight, except in the case of heating
in SDS-containing buffer, and the cleavage efficiencies were
determined by densitometry (NIH Image J software). In
comparison with the result of the use of SDS under harsh
conditions, 74% of the immobilized HSA was recovered with
100 mm NH2OH in the absence of aniline, and the recovery of
immobilized HSA was increased to 88% in the presence of
aniline. The biotin tag was exchanged effectively with the
fluorescent Alexa dye, and 63% HSA–Alexa Fluor 488 was
retrieved. Exchange with the FLAG tag appeared quantita-
tive. Importantly, no detectable levels of HSA were observed
when the beads were incubated with the buffer alone; thus,
the hydrazone is completely stable at pH 6.0.
amine (NH2OH), to give
a more stable oxime (Fig-
ure 2b).[11c,13] The cleavage kinetics depend on the concen-
tration of NH2OH, and a significant, but limited enhancement
was observed upon the addition of 100 mm aniline (see
Figure 4 in the Supporting Information). These results suggest
that at a high concentration of NH2OH (> 10 mm), direct
nucleophilic attack on the CLB354 hydrazone competes with
aniline-catalyzed transimination. At 100 mm NH2OH and
100 mm aniline, cleavage was quantitative within 8 hours (see
Figure 5 in the Supporting Information).
The mechanism of cleavage is expected to be the same in
solution as on (strept)avidin beads. However, the kinetics of
the transimination reaction may be affected by the accumu-
lation of free hydrazine groups on the beads as the reaction
proceeds, and by the local environment created by the
In all studies in which hydrazones have been used as
cleavable functional groups, competing amines have been
used for cleavage.[5–8] As the use of strongly nucleophilic
amines may be incompatible with the function of certain
proteins, we explored an alternative strategy involving the use
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of an aldehyde to capture the hydrazinopyridine group of the
further modification. These results suggest that detailed
analysis of the kinetics and thermodynamics of hydrazones
and oximes could expand the utility of these groups in
chemical bioapplications.
biotin fragment (Figure 1C). In contrast to cleavage with
aminooxy compounds, this strategy requires aniline as a
catalyst for transimination, as the aldehyde itself is electro-
philic and unable to react directly with the hydrazone bond.
For solubility reasons, a PEGylated benzaldehyde was used to
cleave immobilized HSA–CLB354x (x = 1.5) instead of ben-
zaldehyde. Indeed, in the absence of aniline, treatment with
PEGylated benzaldehyde at a concentration of 10 mm over-
night did not result in detectable cleavage of the hydrazone
(Figure 3). However, upon the addition of aniline (100 mm),
transimination occurred, and HSA–benzaldehyde was
retrieved in about 60% yield (Figure 3), whereas PEGylated
biotin remained on the beads. Importantly, in the released
benzaldehyde-functionalized protein, a new synthetic handle
is available for further modification.
Having fully demonstrated the scope of the method in
model systems, we implemented CLB354 in our ongoing
research program aimed at the discovery of new binding
partners of anticoagulant protein S, the known binding
partner of which is complement factor protein C4bP. Recent
studies have suggested that protein S might have several other
functions besides its well-known activated protein C (APC)
cofactor activity.[14–16] Owing to the newly discovered func-
tions of protein S, there has been renewed interest in the
discovery of new binding partners in plasma and on cells.[17]
We synthesized protein S–CLB3543.4 and used it in a pull-
down assay to retrieve its binding partners from human
plasma. Protein S–CLB3543.4 and its binding partners were
pulled down with avidin beads. The beads were washed, and
the remaining proteins were eluted overnight with a solution
at pH 6.0 containing NH2OH (100 mm) and aniline (100 mm).
Approximately 80% of the proteins immobilized on the
beads were eluted by this procedure (see the Supporting
Information). The proteins were analyzed by SDS-PAGE and
western blotting. As anticipated, C4bP was enriched, and a
potential new binding partner of protein S was discovered
(see the Supporting Information).
It is anticipated that the method presented herein is
compatible with a wide range of biomolecules, and the mild
cleavage conditions should enable the retrieval of active,
structurally intact proteins. Indeed, protein S was found to
maintain full APC cofactor activity after incubation overnight
under our harshest cleavage conditions: treatment with
NH2OH (100 mm) and aniline (100 mm) at pH 6.0 (see
Figure 15 in the Supporting Information). Alternatively,
proteins that are incompatible with strong nucleophiles can
be cleaved with an aldehyde in the presence of aniline. The
method is highly flexible and can be optimized to meet the
conditions and requirements of individual systems. Cleavage
times can be reduced significantly by lowering the pH value,
by increasing the temperature, or by increasing the concen-
tration of the nucleophile (aniline, aminooxy groups) in the
cleavage cocktail. For example, the bisaryl hydrazone can be
cleaved quantitatively within 1 h with NH2OH (100 mm) in
0.1m anilinium acetate (pH 4.6; see Figure 8 in the Supporting
Information). The method further distinguishes itself from
existing methods by offering the opportunity to introduce a
new label or affinity tag, or to preserve a synthetic handle for
Received: November 30, 2009
Revised: January 18, 2010
Published online: February 22, 2010
Keywords: affinity purification · bioorganic chemistry ·
.
cleavable linkers · homogeneous catalysis · hydrazones
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